[Show abstract][Hide abstract] ABSTRACT: The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1/V2 region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1/V2 could be partly attributed to selection by vaccine primed T cells. We performed a T-cell based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02 and A*11 restricted epitopes in the MN-strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of post-acquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02(+) compared to A*02(-) participants (VE=54% vs. 3%, p=0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02(+) participants (VE=74% vs. 15%, p=0.02). Additionally, a reanalysis of vaccine-induced immune responses focused on those that were shown to correlate with infection risk, suggested that the humoral response may have differed in A*02(+) participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials.
The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed towards understanding the mechanisms of protection, including this T-cell based sieve analysis which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated post-acquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection.
[Show abstract][Hide abstract] ABSTRACT: The characterization of mixed HIV-1 populations is a key question in clinical and basic research settings. This can be achieved through targeted deep sequencing (TDS), where next-generation sequencing is used to examine in depth a sub-genomic region of interest. This study explores the suitability of IonTorrent PGM(LifeTechnologies) for the TDS-based analysis of HIV-1 evolution. Using laboratory reagents and primary specimens sampled at pre-peak viremia the error rates from misincorporation and in vitro recombination were<0.5%. The sequencing error rate was 2- to 3-fold higher in/around homopolymeric tracts, and could be discerned from true polymorphism using bidirectional sequencing. The limit of detection of complex variants was further lowered by using haplotyping. The application of this system was illustrated on primary samples from an individual infected with HIV-1 followed from pre-peak viremia through six months post-acquisition. TDS provided an augmented view of the extent of genetic diversity, the covariation among polymorphisms, the evolutionary pathways, and the boundaries of the mutational space explored by the viral swarm. Based on its performance, the system can be applied for the characterization of minor viral variants in support of studies of viral evolution, which can inform the rational design of the next generation of vaccines and therapeutics.
Journal of virological methods 05/2014; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A large repository of cryopreserved peripheral blood mononuclear cells (PBMC) samples was created to provide laboratories testing the specimens from Human Immunodeficiency Virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell-Vaccine Immune Monitoring Consortium core repository. The donors included 83 Human Immunodeficiency Virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including Cytomegalovirus, Epstein-Barr virus, Influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) Enzyme Linked ImmunoSpot (ELISpot) and IFN-γ/Interleukin 2 (IL-2) Intracellular Cytokine Staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes an unique resource for laboratories who are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.
Journal of immunological methods 04/2014; · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1-specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor-mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one FCGR2C tag SNP (rs114945036) that associated with VE against HIV-1 subtype CRF01_AE, with lysine at position 169 (169K) in the V2 loop (CRF01_AE 169K). Individuals carrying CC in this SNP had an estimated VE of 15%, while individuals carrying CT or TT exhibited a VE of 91%. Furthermore, the rs114945036 SNP was highly associated with 3 other FCGR2C SNPs (rs138747765, rs78603008, and rs373013207). Env-specific IgG and IgG3 Abs, IgG avidity, and neutralizing Abs inversely correlated with CRF01_AE 169K HIV-1 infection risk in the CT- or TT-carrying vaccine recipients only. These data suggest a potent role of Fc-gamma receptors and Fc-mediated Ab function in conferring protection from transmission risk in the RV144 VE trial.
The Journal of clinical investigation 01/2014; · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The United States (U.S.) military represents a unique population within the human immunodeficiency virus 1 (HIV-1) pandemic. The last comprehensive study of HIV-1 in members of the U.S. Navy and Marine Corps (Sea Services) was completed in 2000, before large-scale combat operations were taking place. Here, we present molecular characterization of HIV-1 from 40 Sea Services personnel who were identified during their seroconversion window and initially classified as HIV-1 negative during screening. Protease/reverse transcriptase (pro/rt) and envelope (env) sequences were obtained from each member of the cohort. Phylogenetic analyses were carried out on these regions to determine relatedness within the cohort and calculate the most recent common ancestor for the related sequences. We identified 39 individuals infected with subtype B and 1 infected with CRF01_AE. Comparison of the pairwise genetic distance of Sea Service sequences and reference sequences in the env and pro/rt regions showed that 5 samples were part of molecular clusters, a group of two and a group of three, confirmed by single genome amplification. Real-time molecular monitoring of new HIV-1 acquisitions in the Sea Services may have a role in facilitating public health interventions at sites where related HIV-1 infections are identified.
AIDS research and human retroviruses 08/2013; · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we explore the association between KIR/HLA and HIV-1 acquisition with different viral subtypes circulating in East Africa. In the prospective CODE cohort (Mbeya,Tanzania) carriers of KIR3DS1 and its putative ligand (HLA-A or HLA-B Bw4-80Ile alleles) showed increased HIV-1 acquisition risk(OR=3.46;95%CI:1.12-10.63;p=0.04) and a trend for enrichment for subtype A and A-containing recombinants (78%vs.46%;OR=4.05;95%CI:0.91-28.30;p=0.09),at the expense of subtype C (11%vs.43%;OR=0.17;95%CI:0.01-0.97;p=0.08).In vitro, only NK cells from KIR3DS1(+)/HLA-Bw4-80Ile(+) healthy donors showed a 2-fold increased capacity to inhibit replication of subtype C vs. subtype A viruses (p=0.01). These findings suggest the presence of an innate sieve effect and may inform HIV-1 vaccine development.
The Journal of Infectious Diseases 08/2013; · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The advent of next generation sequencing technologies is providing new insight into HIV-1 diversity and evolution, which has created the need for bioinformatics tools that could be applied to the characterization of viral quasispecies. Here we present Nautilus, a bioinformatics package for the analysis of HIV-1 targeted deep sequencing data. The DeepHaplo module determines the nucleotide base frequency and read depth at each position and computes the haplotype frequencies based on the linkage among polymorphisms in the same next generation sequence read. The Motifs module computes the frequency of the variants in the setting of their sequence context and mapping orientation, which allows for the validation of polymorphisms and haplotypes when strand bias is suspected. Both modules are accessed through a user-friendly GUI, which runs on Mac OS X (version 10.7.4 or later), and are based on Python, JAVA and R scripts. Nautilus is available from http://www.hivresearch.org.
AIDS research and human retroviruses 06/2013; · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The RV144 HIV-1 vaccine trial (Thailand, 2003-2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrolment in RV144 due to pre-existing HIV-1 infection using a multi-region hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was CRF01_AE: 91.7%, subtype B: 3.5%, B/CRF01_AE recombinants:4.3%, and dual infections: 0.5%. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. 69% of subtype B clustered with cosmopolitan Western B. 93% of B/CRF01_AE recombinants were unique; recombination breakpoints analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52%-68% for CRF01_AE Env, and 12-29% for subtype B immunogens. 43-48% of CRF01_AE sequences differed from the vaccine insert in Env V2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.
[Show abstract][Hide abstract] ABSTRACT: The multi-region hybridization assay (MHAbce) for genotyping HIV-1 subtypes B, C and circulating recombinant form (CRF01_AE) was evaluated on paired plasma and dried blood spots (DBS) collected from 68 HIV-1 infected individuals in Thailand. CRF01_AE was the predominant subtype identified using plasma samples (51/62) and DBS (24/27). There was no discordance in subtype designations between plasma and DBS.
The Southeast Asian journal of tropical medicine and public health 03/2012; 43(2):333-9. · 0.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract The U.S. Army initiated an investigation in response to observations of a possible increase in HIV incidence among soldiers deployed to combat. Human immunodeficiency virus (HIV)-infected U.S. Army soldiers are not eligible to deploy. Combat presents a health hazard to HIV-infected soldiers and they pose a threat to the safety of the battlefield blood supply and their contacts. All soldiers are routinely screened for HIV every 2 years and those who deploy are also screened both prior to and after deployment. Seroconversion rates were estimated for all soldiers who deployed to Afghanistan or Iraq in the period 2001-2007 and all active duty soldiers who did not. Seroconverters with an estimated date of infection, based on calculation of the midpoint between the last seronegative and first seropositive test date, that was either before or during deployment were eligible for inclusion. Confidential interviews and medical record reviews were conducted to determine the most likely time, geographic location, and mode of infection. Reposed predeployment samples were tested for HIV ribonucleic acid. The HIV seroconversion rate among all soldiers who deployed was less than the rate among those who did not deploy: 1.04 and 1.42 per 10,000 person-years, respectively. Among 48 cases, most were determined to have been infected in the United States or Germany and prior to deployment (n=20, 42%) or during rest and relaxation leave (n=13, 27%). Seven seronegative acute infections were identified in the predeployment period. Subtype was determined for 40 individuals; all were subtype B infections. All were acquired through sexual contact. These findings can inform development of preventive interventions and refinement of existing screening policy to further reduce HIV-infected deployed soldier person time.
AIDS research and human retroviruses 01/2012; 28(10):1308-12. · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses.
PLoS ONE 01/2012; 7(4):e29454. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are important innate effector cells controlled by an array of activating and inhibitory receptors. Some alleles of the inhibitory killer-cell immunoglobulin-like receptor KIR3DL1 in combination with its HLA class I ligand Bw4 have been genetically associated with slower HIV-1 disease progression. Here, we observed that the presence of HLA-B Bw4 was associated with elevated frequencies of KIR3DL1(+) CD56(dim) NK cells in chronically HIV-1-infected individuals from the rural district of Kayunga, Uganda. In contrast, levels of KIR2DL1(+) CD56(dim) NK cells were decreased, and levels of KIR2DL3(+) CD56(dim) NK cells were unchanged in infected subjects carrying their respective HLA-C ligands. Furthermore, the size of the KIR3DL1(+) NK cell subset correlated directly with viral load, and this effect occurred only in HLA-B Bw4(+) patients, suggesting that these cells expand in response to viral replication but may have relatively poor antiviral capacity. In contrast, no association with viral load was present for KIR2DL1(+) and KIR2DL3(+) NK cells. Interestingly, chronic HIV-1 infection was associated with an increased polyfunctional response in the NK cell compartment, and, upon further investigation, KIR3DL1(+) CD56(dim) NK cells exhibited a significantly increased functional response in the patients carrying HLA-B Bw4. These results indicate that chronic HIV-1 infection is associated with increased NK cell polyfunctionality and elevated levels of KIR3DL1(+) NK cells in Ugandans carrying the HLA-B Bw4 motif.
Journal of Virology 03/2011; 85(10):4802-11. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4(+) T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4(+) T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.
Journal of Virology 02/2011; 85(4):1541-53. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dual infection with diverse HIV strains can foster the emergence of recombinants. The resulting increase in viral genetic diversity is a major challenge for vaccine development HIV treatment. In this study we aim to investigate the socio demographic factors associated with an increasing level of genetic diversity among HIV strains in a population of drug-users in Northern Thailand.
From 1999 through 2000, 2231 volunteers were enrolled in the Opiate-Users Research in Chiang Mai, Thailand. HIV subtype analysis was conducted among those HIV-1 seropositive (n=347) using a multi-region hybridization assay. Social and demographic variables were assessed using a structured questionnaire.
Overall, 336/347 (96.8%) of the samples could be typed. 81.8% were CRF01_AE, 3.9% were subtype B, 9.2% were recombinants (mostly between CRF01_AE and B) and 5.1% were dual infections. Dual infections were more frequent among those with a lower education level (AOR: 5.2; 95% CI 1.4-20.3), those who have initiated injecting in the last 3 years (AOR: 3.9; 95% CI 1.1-14.6), and those reporting frequent needle sharing in the last 3 months (AOR: 7.0; 95% CI 1.5-34.1). Both recombinant strains and dual infection were more frequent among those reporting frequent needle sharing in the last 3 months (AOR: 5.3; 95% CI 1.6-17.1).
To limit the expanding complexity of HIV-1 strains, early intervention should be aimed at reduction in needle sharing, especially among new intravenous drug users.
Drug and alcohol dependence 12/2010; 116(1-3):24-30. · 3.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crucial role of recombination in HIV-1 biology is being increasingly recognized. In vitro studies have shown that up to 30 strand-transfer events may occur per viral replication cycle. Thus, recombination may surpass mutation as a major mechanism driving HIV-1 evolution. Currently, recombinant strains comprise 37% of the full-genome HIV-1 sequence database, including sequences representing 47 Circulating Recombinant Forms (CRFs) and more than 250 different Unique Recombinant Forms (URFs). Mapping of recombination breakpoints helps establish relationships among strains that are related by descent, such as CRF07_BC and CRF08_BC in China, and sheds light on their origin and epidemic spread. Additionally, unrelated recombinants sharing common breakpoints may reflect recombination hotspots within the viral genome. Here we present a software tool, RecDraw, for the graphical representation and efficient comparison of recombinant HIV-1 structures and breakpoints. RecDraw is a platform-flexible, Java stand-alone application available through http://www.hivresearch.org/research.php?ServiceID = 5&SubServiceID = 6 .
AIDS research and human retroviruses 10/2010; 26(12):1317-21. · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we explore associations between HLA variation and human immunodeficiency virus type 1 (HIV-1) acquisition and disease progression in a community cohort in Mbeya, Tanzania, a region that, despite harboring high rates of HIV-1 infection, remains understudied. African-specific allele HLA-A*74:01 was associated with decreased risk of infection (odds ratio [OR], 0.37; 95% confidence interval [CI], 0.14-0.80; P = .011) and with protection from CD4(+) cell counts <200 cells/uL in women (OR, 0.31; 95% CI, 0.07-0.91; P = .032) and men (OR, 0.15; 95% CI, 0.01-0.78; P = .020). These associations remained significant after adjustment for linkage disequilibrium with HLA-B and HLA-C alleles. This observation calls for additional investigation of mechanisms by which HLA-A*74:01 may influence HIV-1 acquisition and control of the infection.
The Journal of Infectious Diseases 10/2010; 202(10):1562-6. · 5.85 Impact Factor